Abstract
Acute myocardial infarction is mainly caused by a lack of blood flood in the coronary artery. Angiopoietin-like protein 2 (ANGPTL2) induces platelet activation and thrombus formation in vitro through binding with immunoglobulin-like receptor B, an immunoglobulin superfamily receptor. However, the mechanism by which it regulates platelet function in vivo remains unclear. In this study, we investigated the role of ANGPTL2 during thrombosis in relationship with ST-segment elevation myocardial infarction (STEMI) with spontaneous recanalization (SR). In a cohort of 276 male and female patients, we measured plasma ANGPTL2 protein levels. Using male Angptl2-knockout and wild-type mice, we examined the inhibitory effect of Angptl2 on thrombosis and platelet activation both in vivo and ex vivo. We found that plasma and platelet ANGPTL2 levels were elevated in patients with STEMI with SR compared to those in non-SR (NSR) patients, and was an independent predictor of SR. Angptl2 deficiency accelerated mesenteric artery thrombosis induced by FeCl3 in Angptl2–/– compared to WT animals, promoted platelet granule secretion and aggregation induced by thrombin and collogen while purified ANGPTL2 protein supplementation reversed collagen-induced platelet aggregation. Angptl2 deficiency also increased platelet spreading on immobilized fibrinogen and clot contraction. In collagen-stimulated Angptl2–/– platelets, Src homology region 2 domain–containing phosphatase (Shp)1-Y564 and Shp2-Y580 phosphorylation were attenuated while Src, Syk, and Phospholipase Cγ2 (PLCγ2) phosphorylation increased. Our results demonstrate that ANGPTL2 negatively regulated thrombus formation by activating ITIM which can suppress ITAM signaling pathway. This new knowledge provides a new perspective for designing future antiplatelet aggregation therapies.
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Background
Myocardial infarction is caused by partial or complete blockade of a coronary artery and is a global health challenge with high incidences of morbidity and mortality [1, 2]. ST-segment elevation myocardial infarction (STEMI) is prevalent and is typically caused by aberrant thrombosis leading to vascular obstruction. Primary percutaneous coronary intervention (PPCI) is the standard of care for treating STEMI in order to restore blood flow in the infarct region [3]. Approximately, 15–18% of patients with STEMI undergo favorable spontaneous recanalization (SR) of occluded vessel because of thrombus dissolution prior to PPCI [4,5,6]. These SR patients recover better than those with persistent occlusion of the arteries [6] and SR is considered to be an independent predictor of favorable outcome in patients with STEMI [5]. Balance between pro-thrombotic and antithrombotic signals play a crucial role in stopping a bleed without causing intravascular thrombosis [5]. Thrombosis is a highly complex process that involves platelets, vessels, and the coagulation system, in which platelets play a key role [7, 8]. Pro-activation and inhibition signals are also present in platelets [8, 9]. Therefore, a better understanding of platelets is of great value for clinical antithrombotic therapy.
Our previous study found that paired immunoglobulin-like receptor B (PIRB), the mouse orthologue of human LILRB2, which contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), inhibits platelet activation pathway initiated by the immunoreceptor tyrosine-based activation motif (ITAM) via the activation of Src homology region 2 domain-containing phosphatases 1/2 (SHP1/2) [9]. As the ligand of PIRB, angiopoietin-like protein (ANGPTL)2, is expressed and stored in platelet α-granules [9]. ANGPTL2 was first discovered and cloned in 1999. It is one of seven members of the ANGPTL protein family. Functionally, ANGPTL2 possesses proinflammatory properties and plays a role in the development of a number of chronic diseases, including cancer, diabetes, and atherosclerosis [10,11,12]. In vitro, purified ANGPTL2 protein inhibited platelet aggregation stimulated by various agonists, such as collagen, thrombin, and adenosine diphosphate (ADP) through its receptor PIRB [9]. However, it remains unclear whether and how ANGPTL2 influences platelet aggregation and thrombosis in vivo.
The objective of this study was to explore the expression of ANGPTL2 in STEMI patients with SR and the role of ANGPTL2 in arterial thrombus formation, and to clarify the possible mechanisms. This will provide new target for the prevention and therapy of thrombosis.
Materials and methods
Study design
Patients who developed STEMI and underwent PPCI between August 2016 and November 2019 at the Shanghai Ninth People’s Hospital, affiliated to the Shanghai Jiao Tong University School of Medicine, were recruited in this study. Patients were excluded if they had a history of coronary heart disease, late presentation (> 12 h), infectious or inflammatory disease, severe liver or renal disease or thrombolytic therapy. The STEMI diagnosis was confirmed by coronary angiography, which was followed by the administration of 300 mg aspirin, 180 mg ticagrelor, and 0.15 g·kg-1·min-1 tirofiban infusion. On the basis of the thrombolysis in myocardial infarction (TIMI) flow grade, patients who underwent PPCI were divided into two subgroups: the SR group and non-SR (NSR) group. The observational clinical study complied with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations [13]. All patients provided informed written consent prior to sample collection, and the study protocol was approved by the Shanghai Ninth People’s Hospital Institutional Ethics Committee (HKDL2017300) (No. 2016-256-T191) and performed in accordance with the ethical standards outlined in the 1964 World Medical Association Declaration of Helsinki.
Plasma preparation and ANGPTL2 quantification
After informed consent was obtained, blood was collected from the patients into plasma sodium citrate anticoagulant tube prior to medication. Plasma fraction was obtained by differential centrifugation at 3000 rpm for 10 min at 26 ℃. ANGPTL2 levels were measured using an enzyme-linked immunosorbent assay kit (E1919Hu, USCN Life Science Inc., Wuhan, China) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using an Infinite M200PRO microplate reader (Tecan, Switzerland, Germany).
Platelet preparation and measurement of platelet ANGPTL2 levels
Blood was collected from patients into empty syringes moistened with sodium citrate and transferred into polypropylene centrifuge tubes containing 100 μL/mL White’s anticoagulant (2.94% sodium citrate, 136 mM glucose, pH 6.4), 0.1 g/mL PGE1, 1 U/mL apyrase and gently mixed. Platelet-rich plasma (PRP) was fractionated using differential centrifugation (290 g for 10 min). Supernatant was aspirated and the platelets were resuspended with PRP containing 5 mM EDTA and subjected to second differential centrifugation (850 g for 10 min). Washed platelets were resuspended in the modified Tyrode’s buffer as previously described [14].
Platelet ANGPTL2 levels were evaluated immunoblotting as previously described with goat anti-ANGPTL2 antibody (1:1000, AF2084, R&D Systems, USA) and the secondary antibodies (1:3000, #L3042, Signalway Antibody, China) [9]. Image Pro Plus v6.0 (Media Cybernetics, USA) was used to quantify specific protein bands and to determine ANGPTL2 expression levels in platelets. Then data were normalized using the ratio of the band gray value of ANGPTL2 to GAPDH.
Generation of Angptl2–/– mice
All animal experiments were approved by the Shanghai Ninth People’s Hospital institutional ethics committee (HKDL2017300). Global Angptl2 deficient (Angptl2–/–) mice were obtained from Jackson Laboratory, while wild-type (WT) C57BL/6 mice genetic background control mice were obtained from Shanghai SLAC Experimental Animal Co., Ltd. Angptl2 deficiency in platelets was confirmed by comparing western blots of platelet extracts of Angptl2−/− and wild-type (WT) C57BL/6 J mice. All mice were maintained in the Division of Laboratory Animal Resources under specific pathogen free conditions. After the mice were bred, animals homozygous for the null allele were screened and identified. Mice were euthanized with an overdose of isoflurane anesthesia.
In vivo thrombosis models
Male WT and Angptl2−/−mice with a C57BL/6 J background (age, 4–5 weeks old) were used to establish the ferric-chloride (FeCl3)-induced mesenteric arteriole injury model. The mice were anesthetized with a ketamine/xylazine mixture (200:10 mg·kg−1) via jugular vein cannulation. Rhodamine dye to label platelets was also delivered via this catheter. After the mesentery was exteriorized through a midline abdominal incision, arterioles with diameters of 80–100 μm were treated with filter paper pre-saturated with 250 mM FeCl3 for 5 min. The mesenteric arterioles and thrombus formation were visualized using an AxioPlan 2 microscope equipped with an AxioCam MRm camera (Carl Zeiss, Göttingen, Germany). Z-STACK slices of the vessel were captured. Thrombus volumes were determined from the area and height of the clot after the rendered z-stack images were deconvolved with AxioVision Rel 4.6 software (Carl Zeiss, Göttingen, Germany) to produce a three-dimensional image. Thrombus stability was determined by calculating the percentage of the vessel occupied by the thrombus within 2 min and quantified using a score from 1 to 10, with 1 representing 0% to 10% occupancy and 10 representing 91% to 100% occupancy [15, 16]. In these experiments, three mice per group, with 10 thrombi per mouse, were studied.
Platelet secretion assay
Fresh blood was collected from the abdominal aorta of mice anesthetized via isoflurane inhalation, with syringes containing 100 mL/L sodium citrate anticoagulant (2.94% sodium citrate, 136 mM glucose [pH 6.4]), 0.1 g/mL prostaglandin E1, and 1 U/mL apyrase. Washed platelets were prepared following the above method. The count was read using an automatic blood cell analyzer (BM860, BWLIN MAN, China) and adjusted to 1 × 106 platelets/µL with a modified Tyrode’s buffer.
Platelet secretion was analyzed by measuring the surface expression of α (P-selectin)-and dense-granule, markers of granule release. For α-granule analysis, platelets from WT and Angptl2−/− mice were stained with a fluorescein isothiocyanate-conjugated monoclonal antibody targeting P-selectin (Biolegend, #148305). For dense-granule exocytosis analysis [17], 100 μL of washed platelets (2 × 107/mL) were incubated with 4 μM fluorescent quinacrine (Q3251, Sigma-Aldrich) at 37 ℃ for 10 min. After stimulated by collagen (#385, Chrono-Log), thrombin (T4648, Sigma-Aldrich, USA) or adenosine diphosphate (ADP; A5258, Sigma-Aldrich), platelet secretion was analyzed by flow cytometry using CytoFLEX (Beckman Coulter, USA).
Platelet aggregation
Then, the washed platelets (300 µL) were used for an aggregation assay in a Lumi-Aggregometer (Chrono-Log, Havertown, PA, USA) [9]. The role played by Angptl2 in agonist-induced platelet aggregation was investigated by stimulating platelets from WT and Angptl2−/− mice with collagen, thrombin or ADP. For further analysis, purified ANGPTL2 was added at concentrations of 0.1, 0.5, and 1 μg/mL to washed WT platelets stimulated with 1 μg/mL collagen, 0.067 U/mL thrombin, or 1 μM/mL ADP. The effect of ANGPTL2 was determined on the basis of decrease in platelet aggregation.
Platelet spreading on immobilized fibrinogen
Chamber slides with microtiter wells (Nalge Nunc, Rochester, NY, USA) were coated with 50 µg/mL human fibrinogen (Sigma-Aldrich) and incubated at 4 °C overnight. Washed mouse platelets (2 × 107/mL) were stored in Tyrode’s buffer containing 1 mM CaCl2 and 1 mM MgCl2, and incubated at 37 °C for 90 min. Subsequently, the washed platelets were stimulated with 0.01 U/mL thrombin, and spread on immobilized fibrinogen for 60 min. Then, they were fixed, permeabilized, stained with fluorescein-labeled phalloidin (Invitrogen, Carlsbad, CA, USA), and visualized using an upright fluorescence Axio Scope.A1 microscope (Carl Zeiss, Göttingen, Germany) equipped with a 100 × /1.30 oil objective lens, an X-Cite 120Q light source (EXFO, Mississauga, CA, USA), and a digital camera. At least six images were randomly chosen for each experiment and analyzed under blinded conditions. The platelet surface area was analyzed using Image J software v1.8.0 (National Institutes of Health, Bethesda, MD, USA) [9].
Platelet-mediated clot retraction
Washed mouse platelets were mixed with citrated normal human platelet-depleted plasma to a concentration of 3 × 108/mL. Subsequently, coagulation was induced using 0.5 U/mL thrombin. The clots were allowed to retract at 37 °C and then photographed at 60 min. Two-dimensional clot size was quantified from photographs by using Image J software v1.8.0, and clot retraction was expressed in terms of the retraction ratio (1–[final clot size/initial clot size]) [9].
Western blot analysis
Western blot was performed as previously described [9]. Platelets (3 × 108/mL) from WT and Angptl2–/– mice were stimulated with 1 μg/mL collagen at 37 °C. After the platelets were treated with collagen for 1, 2, 3, 4, or 5 min, they were lysed with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) on ice for 30 min. Subsequently, they were centrifuged at 13,000×g for 10 min at 4 °C to remove cell debris, following which the protein supernatant was stored in a new tube for western blot analysis. The target protein levels were determined using anti-Angptl2 polyclonal antibody (1:1000, #AF 1444, R&D, USA), the secondary antibodies (1:3000, #L3042, Signalway Antibody, China), anti-phospho-Shp1 Y564 (1:1000, #8849, Cell Signaling Technology,USA), anti-phospho-Shp2 Y580 (1:1000, #5431, Cell Signaling Technology, USA), anti-phospho-Src Y416 (1:1s000, #6943, Cell Signaling Technology, USA), anti-phospho Syk Y525 (1:1000, #PA5-104904, Invitrogen, USA), anti-phospho-PLCγ2 Y1217 (1:1000, #3871, Cell Signaling Technology, USA), anti-GAPDH antibody (1:1000, #5174, Cell Signaling Technology, USA) and the secondary antibodies (1:3000, #L3012, Signalway Antibody, China). GAPDH was used as a loading control for total protein.
Statistical analysis
Statistical analysis was performed using SPSS (v.21.0; IBM Corp., Armonk, NY, USA). Quantitative data were presented as the mean ± standard deviation (SD). Continuous variables were tested for normality by using the Kolmogorov–Smirnov test. The chi-square test was used for categorical variables. Multivariate logistic regression analysis was used to determine the independent predictors of SR. Statistical significance was determined via an independent sample t-test between two groups or ANOVA among multiple groups using SPSS (v.21.0). Statistical significance was set at P < 0.05.
Results
ANGPTL2 level was correlated with SR in patients with STEMI
A total of 276 STEMI patients (229 male, 47 female, mean age 64.46 ± 10.66 years old) were included, with 108 SR and 168 NSR patients. The clinical characteristics were shown in Table 1. No significant differences in age, sex and blood pressure were found between SR and NSR groups. However, plasma ANGPTL2 levels in the SR group were significantly higher (P < 0.001) than those in the NSR group (Fig. 1A). Multiple logistic regression analysis was therefore performed to determine the independent predictors of SR in patients with STEMI. The plasma ANGPTL2 level (95% confidence interval [CI] 0.828–0.912, P < 0.001), uric acid concentration (95% CI 1.005–1.015, P < 0.001), and mean platelet volume (MPV) (95% CI 1.773–4.056, P < 0.001) were found to be independent predictors of SR Table 2. In resting platelets, ANGPTL2 is expressed and stored in α-granules [9], and when platelets are activated, ANGPTL2 is released and bound to the surface of platelets [9], so platelet ANGPTL2 expression was also measured in patients from both groups using western blot analysis. ANGPTL2 levels were significantly higher in patients with SR (n = 5) than those in patients without SR (n = 5; P < 0.01; Fig. 1B).
Angptl2 –/– mice exhibited normal hematopoiesis
To evaluate the role of Angptl2 in vivo, we generated Angptl2–/– mice. To ensure loss of Angptl2 does not affect the hematopoietic system, peripheral blood samples of eight Angptl2–/– mice and eight sex- and age-matched WT mice were collected and analyzed. As shown in Table 3, we did not observe any alterations in the hematopoietic system including hemoglobin (Hb) level, white blood cell (WBC), red blood cell (RBC), and platelet counts between Angptl2–/–and WT animals (n = 8; P > 0.05; Table 3).
Angptl2 –/– mice displayed enhanced mesenteric artery thrombosis induced by FeCl3 in vivo
To evaluate if Angptl2 participates in thrombus growth, we subjected Angptl2–/–and WT animals to FeCl3-induced mesenteric arteriole injury and monitored platelets by fluorescent labeling. Under normal conditions, immediate after vascular endothelium injury platelets rapidly (within 10 min) accumulated as puncta and merge into large thrombus at the injured sites. A total of 29 thrombi selected from 3 WT mice and 30 thrombi selected from 3 Angptl2–/– mice were evaluated (9–10 per mouse). The thrombus in the injured arterioles of Angptl2–/– mice developed faster and were substantially larger than those in WT mice (Fig. 2A and B). The volume of the final thrombus was significantly greater in Angptl2–/– mice compared to WT mice (Angptl2–/–: 21,5529.83 ± 56718.75 μm3; WT 149,703.45 ± 25971.98 μm3; P < 0.001; Fig. 2C). Furthermore, the thrombus was more stable in Angptl2–/– mice than that in WT mice (stability score: 6.85 ± 1.34 vs 2.27 ± 0.57; P < 0.001; Fig. 2D).
Loss of Angptl2 exacerbated platelet α-and dense-granule secretion
To further clarify the role of Angptl2 in platelet activation and thrombosis, we isolated platelets from Angptl2–/– mice for further characterization. First, by immunoblotting assay we confirmed the loss of Angptl2 protein expression in Angptl2–/– platelets (Fig. 3A). Next, we asked if loss of Angplt2 protein affected platelet activation by measuring α (P-selectin)-and dense-granule secretion after collagen, thrombin, or ADP stimulation. Compared to WT platelets, Angptl2–/– platelets released more α and dense granules when stimulated with collagen and thrombin (n = 6; P < 0.001) (Fig. 3B, C). However, we observed no difference in α- and dense-granule secretion upon ADP stimulation (Fig. 3D), suggesting Angptl2 mostly responds downstream of thrombin and collagen stimulation.
Loss of Angptl2 increased platelet aggregation in vitro
Next, we asked if Angplt2 participated in platelet aggregation. To accomplish this, isolated platelets were stimulated with either collagen, thrombin, or ADP and degree of platelet aggregation was evaluated. Angptl2 deficiency resulted in enhanced collagen (n = 6; P < 0.001)-or thrombin (n = 6; P < 0.01)-induced platelet aggregation compared to WT (Fig. 4A). Similar to α- and dense-granule secretion results, ADP-stimulated platelet aggregation did not differ between WT and Angptl2–/– platelets (Fig. 4A). Together, these results indicated that Angptl2 inhibited collagen- and thrombin-induced platelet aggregation.
To further analyze the inhibitory role of Angptl2, purified ANGPTL2 protein was reintroduced to WT platelets at 0.5 or 1 ug/mL concentrations that were stimulated with collagen, thrombin, or ADP and platelet aggregation was evaluated. Addition of ANGPTL2 inhibited collagen-induced platelet aggregation in a dose-dependent manner (n = 6; P < 0.001) (Fig. 4B). Surprisingly, addition of ANGPTL2 protein did not block thrombin-or ADP-induced platelet aggregation (Fig. 4C, D). The findings suggested that ANGPTL2 inhibited platelet aggregation mainly through the collagen-stimulated signaling pathway.
Loss of Angptl2 promoted platelet spreading on immobilized fibrinogen and clot contraction
The binding activity of WT and Angptl2–/– platelets was evaluated using their static adhesion to immobilized fibrinogen. Angptl2–/– platelets were found to spread more extensively than WT platelets on immobilized fibrinogen (average area covered in 60 min: WT, 971.17 ± 74.50 pixels vs. Angptl2–/–, 1285.18 ± 49.34 pixels; n = 6; P < 0.001) (Fig. 5A and B). In addition, Angptl2 is related to clot retraction driven by integrin αIIbβ3. The average clot retraction rate in PRP samples containing WT and Angptl2–/– platelets were 0.29 ± 0.01 and 0.38 ± 0.02 (n = 6; P < 0.001), respectively, at 60 min (Fig. 5C and D), suggesting that clot retraction in PRP was accelerated in Angptl2–/– platelets.
Angptl2 regulates ITIM and GPVI-ITAM signaling
To elucidate the mechanisms Angptl2 mediating platelet activity, tyrosine phosphorylation during ITIM and ITAM signaling was measured in WT and Angptl2–/– mouse platelets stimulated with 10 μg/mL collagen for 5 min. The levels of Shp1-Y564 and Shp2-Y580 phosphorylation in ITIM signaling gradually increased with prolonged stimulation in WT platelets. In contrast, phosphorylation was significantly lower in Angptl2–/– mice (P < 0.01) (Fig. 6A–C).
To determine the role of Angptl2 in ITAM signaling, tyrosine phosphorylation was examined using platelet collagen receptor glycoprotein VI (GPVI)-mediated signaling molecules Src, Syk, and phospholipase Cγ2 (PLCγ2). In WT platelets, the levels of tyrosine phosphorylation of Src, Syk, and PLCγ2 increased from 1 to 3 min, and steadily decreased after that. In Angptl2–/– platelets, the levels of tyrosine phosphorylation of Src, Syk, and PLCγ2 were greater than those observed in WT platelets at each detection time point (P < 0.01) (Fig. 6D–F). Src, Syk, and PLCγ2 expression levels did not significantly differ in WT and Angptl2–/– platelets.
Discussion
The major findings of this study are as follows: (1) plasma and platelet ANGPTL2 levels were elevated in patients with STEMI with SR of the infarction-related artery compared to those in non-SR patients, and were an independent predictor of SR; (2) Angptl2 knock out accelerated mesenteric artery thrombosis induced by FeCl3 in vivo, and the thrombi were more stable in Angptl2−/− mice than those in WT mice; (3) Angptl2 deficiency promoted platelet granule secretion and aggregation induced by collagen and thrombin, and purified ANGPTL2 protein reversed the collagen-induced; (4) Angptl2 deficiency promoted platelet spreading on immobilized fibrinogen and clot contraction; and (5) Angptl2 deficiency inhibited ITIM signaling pathways and activated GPVI-ITAM signaling.
Thrombosis is a highly complicated pathological process in which the vasculature, platelets, and coagulation system interact with each other [7, 8]. Platelet adhesion and aggregation play a key role in this process [7, 8]. Mammalian platelets are mainly derived from bone marrow megakaryocytes. They contain a variety of receptors on their surface, including immunoglobulin receptors, a large group of cell surface proteins that belong to the immunoglobulin superfamily and are involved in cell adhesion, binding, and recognition [18,19,20]. Many immunoglobulin superfamily members on the platelet surface participate in platelet adhesion, activation, and aggregation [9, 19, 20]. As a typical member of the immunoglobulin superfamily, GPVI has a short cytoplasmic structural domain and lacks a signaling motif [9, 20]. However, it can bind to the ITAM of the Fc receptor γ chain to form a GPVI/FcR γ chain complex, which potently activates αIIbβ3 and causes platelet aggregation, playing an important role in hemostasis and thrombosis [9, 20]. In addition to ITAM-containing receptors, there are many ITIM-containing immunoglobulin receptors on platelet surfaces, such as platelet endothelial cell adhesion molecule-1 (PECAM1) [21] and paired immunoglobulin-like receptor B (PIRB) [9]. With six extracellular Ig structural domains and a cytoplasmic ITIM, PECAM1 weakly inhibits human or mouse platelet activation induced by collagen, ADP, or thrombin [21]. Mouse PIRB, a homolog of human LILRB2, is also an ITIM-containing immunoglobulin receptor [9, 22]. According to our previous findings, PIRB and LILRB2 are expressed in mouse and human platelets, respectively [9]. PIRB contains six extracellular immunoglobulin domains and four cytoplasmic ITIMs, and mutations in Pirb upregulate platelet activation in mice by suppressing activation of the ITIM signaling pathway and subsequently decreasing recruitment of Shp1 and Shp2 phosphatases [9].
According to the accepted theory [23,24,25,26,27,28,29,30,31,32], upon activation by collagen, platelet GPVI translocates into lipid rafts [23], where Src family kinases (SFKs), including Src, Fyn and Lyn [24], bind to the conserved proline-rich region PxxP in GPVI and become activated by phosphorylation [25,26,27,28,29]. Activated SFKs phosphorylate ITAM in the cytoplasmic region of the GPVI/FcR γ chain, which then recruits and phosphorylates splenic tyrosine kinase (Syk) [25,26,27,28,29,30]. Activated Syk binds to ITAM tyrosine via a tandem SH2 structural domain. Subsequently, the signal is propagated by the activated Syk that phosphorylates the linker for activation of T-cells (LAT). LAT is a transmembrane scaffolding protein with many tyrosine residues. Phosphorylated LAT binds different kinases, such as PI3K (p85/p110), through the SH2 structural domain of the p85 subunit and junctional molecule grb-2-associated binding protein-1 (Gab1) to form a multiprotein complex that activates PLCγ2 [31, 32]. The subsequent rise in the intracellular calcium concentration augments secretion and increases affinity of integrin αIIbβ3, promoting fibrinogen binding, platelet aggregation, and thrombosis [32]. Then, the secondary agonists released from activated platelets, including ADP, thromboxane A2, and 5-hydroxytryptamine, bind to G protein-coupled receptors to activate surrounding resting platelets, synergistically enhancing the response [26]. In addition to phosphorylating ITAMs, SFKs also activate ITIM-containing receptors by phosphorylating ITIM tyrosine residues, which serve as binding sites for the SH2 structural domain of phosphatases such as tyrosine phosphatase SHP1/SHP2 and inositol phosphatase SHIP1/SHIP2, leading to the inactivation of ITAM signaling pathway components [33]. Furthermore, binding of a protein to an ITIM-containing receptor can also isolate the protein from its site of action: for example, the recruitment of the PECAM-1 receptor for p85 prevents p85 translocation into lipid rafts, which reduces PI3K attachment to Gab1 and LAT in lipid rafts and thereby diminishes activation of the PI3K signaling pathway [26, 32,33,34,35,36,37,38]. All of these can lead to the inhibition of platelet activation and thrombosis.
Our previous study found that ANGPTL2, a ligand for the ITIM-containing receptors PIRB and LILRB2, was expressed and stored in platelet α-granules [9]. Moreover, purified ANGPTL2 protein inhibited agonist-induced platelet aggregation and spreading on fibrinogen by binding to PIRB and suppressing collagen receptor GPVI and integrin aIIbb3-mediated signaling [9].
In the present study, we found that plasma and platelet ANGPTL2 levels were elevated in patients with STEMI with SR compared to those without SR. Furthermore, ANGPTL2 level was an independent predictor of SR. Consistent with the clinical data, endogenous ANGPTL2 deficiency enhanced mesenteric artery thrombosis induced by FeCl3 and improved thrombus stability in mice. These results suggested that ANGPTL2 levels are inversely related to thrombus stability, and that plasma and platelet ANGPTL2 may serve as a clinical predictor of autolytic recanalization. This finding is significant as it may guide the prognosis of clinical patients with acute myocardial infarction and adjustment of antithrombotic drug applications.
To clarify the mechanisms of endogenous ANGPTL2 effects on platelet activation, Angptl2−/− mice were used. In agreement with our previously reported in vitro observations of the inhibition of platelet activation by purified ANGPTL2, deficiency of endogenous ANGPTL2 increased collagen- and thrombin-induced platelet activation. Granule secretion and aggregation, as well as platelet spreading on immobilized fibrinogen and clot contraction were all elevated by endogenous ANGPTL2 deficiency. However, evidence from platelet aggregation tests suggested that purified ANGPTL2 protein reversed only the collagen-induced platelet activation, demonstrating that ANGPTL2 has a key inhibitory role in collagen-induced platelet activation. In contrast, the mechanism of the thrombin-induced platelet activation is much more complex, and ANGPTL2 is apparently not the most critical regulator of this process.
To explore the mechanisms of ANGPTL2 effects in more detail, we examined changes in the signaling pathway that involves PIRB/LILRB2, which is the receptor for ANGPTL2. In particular, we investigated PIRB/LILRB2-induced ITIM activation and the resulting ITAM signaling pathway inhibition [9, 26, 39, 40]. Western blot experiments showed that ANGPTL2 deficiency led to decreased phosphorylation of SHP1 and SHP2 in the ITIM signaling pathway, as well as increased p-Src (Y416), p-PLCr2 (Y1217), and p-Syk (Y525) expression, indicating that ANGPTL2 deletion inhibited ITIM that blocks the ITAM signaling pathway. Therefore, increased activation of ITAM signaling pathways resulted in more platelet activation and thrombosis.
Conclusions
Based on the above evidence, we concluded that by binding to the PIRB/LILRB2 receptor and activating the ITIM signaling pathway which inhibit the ITAM signaling pathway, endogenous ANGPTL2 inhibited platelet activation and thrombosis, and reduced thrombi stability. In conclusion, elevated ANGPTL2 expression in plasma and platelets promoted SR of obstructed coronary arteries in patients with acute myocardial infarction. In addition, ANGPTL2 expression level was an independent predictor of SR. The antithrombotic effect of ANGTPL2 provides a new perspective for clinical antiplatelet aggregation therapy. However, the study has some limitations. First, the clinical trial in this study did not carry out long-term follow-up of the included patients, which could not comprehensively analyze the impact of ANGPTL2 on the long-term prognosis of patients. Second, the in vivo thrombosis assay was performed using Angptl2 systemic knockout mice rather than platelet-specific knockout mice, which could not avoid the potential effects of a systemic Angptl2-knockout. In the future, we plan to design more comprehensive clinical trials and use platelet A2-specific knockout mice to better elucidate the role and mechanism of ANGPTL2 in thrombosis.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- STEMI:
-
ST-segment elevation myocardial infarction
- PPCI:
-
Primary percutaneous coronary intervention
- SR:
-
Spontaneous recanalization
- PIRB:
-
Paired immunoglobulin-like receptor B
- ITIMs:
-
Immunoreceptor tyrosine-based inhibition motifs
- ITAM:
-
Immunoreceptor tyrosine-based activation motif
- SHP1/2:
-
Src homology region 2 domain-containing phosphatases 1/2
- ANGPTL:
-
Angiopoietin-like protein
- ADP:
-
Adenosine diphosphate
- TIMI:
-
Thrombolysis in myocardial infarction
- NSR:
-
Non-SR
- STROBE:
-
Strengthening the Reporting of Observational Studies in Epidemiology
- PRP:
-
Platelet-rich plasma
- WT:
-
Wild-type
- FeCl3 :
-
Ferric-chloride
- SD:
-
Standard deviation
- Hb:
-
Hemoglobin
- WBC:
-
White blood cell
- RBC:
-
Red blood cell
- GPVI:
-
Glycoprotein VI
- PLCγ2:
-
Phospholipase Cγ2
- PECAM1:
-
Platelet endothelial cell adhesion molecule-1
- PIRB:
-
Paired immunoglobulin-like receptor B
- SFKs:
-
Src family kinases
- Syk:
-
Splenic tyrosine kinase
- LAT:
-
Linker for activation of T-cells
- Gab1:
-
Grb-2-associated binding protein-1
References
Tsao CW, Aday AW, Almarzooq ZI, Anderson CAM, Arora P, Avery CL et al (2023) Heart disease and stroke statistics-2023 update: a report from the American Heart Association. Circulation 147(8):e93–e621
Tsao CW, Aday AW, Almarzooq ZI, Alonso A, Beaton AZ, Bittencourt MS et al (2022) Heart disease and stroke statistics-2022 update: a report from the American Heart Association. Circulation 145(8):e153–e639
Keeley EC, Boura JA, Grines CL (2003) Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet 361(9351):13–20
DeWood MA, Spores J, Notske R, Mouser LT, Burroughs R, Golden MS et al (1980) Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med 303(16):897–902
Huisse MG, Lanoy E, Tcheche D, Feldman LJ, Bezeaud A, Anglès-Cano E et al (2007) Prothrombotic markers and early spontaneous recanalization in ST-segment elevation myocardial infarction. Thromb Haemost 98(2):420–426
Fefer P, Beigel R, Atar S, Aronson D, Pollak A, Zahger D et al (2017) Outcomes of patients presenting with clinical indices of spontaneous reperfusion in ST-elevation acute coronary syndrome undergoing deferred angiography. J Am Heart Assoc 6(7):e004552
Furie B, Furie BC (2008) Mechanisms of thrombus formation. N Engl J Med 359(9):938–949
Bye AP, Unsworth AJ, Gibbins JM (2016) Platelet signaling: a complex interplay between inhibitory and activatory networks. J Thromb Haemost 14(5):918–930
Fan X, Shi P, Dai J, Lu Y, Chen X, Liu X et al (2014) Paired immunoglobulin-like receptor B regulates platelet activation. Blood 124(15):2421–2430
Kim I, Moon SO, Koh KN, Kim H, Uhm CS, Kwak HJ et al (1999) Molecular cloning, expression, and characterization of angiopoietin-related protein: angiopoietin-related protein induces endothelial cell sprouting. J Biol Chem 274(37):26523–26528
Thorin-Trescases N, Labbé P, Mury P, Lambert M, Thorin E (2021) Angptl2 is a marker of cellular senescence: the physiological and pathophysiological impact of Angptl2-related senescence. Int J Mol Sci 22(22):12232
Deng M, Lu Z, Zheng J, Wan X, Chen X, Hirayasu K et al (2014) A motif in LILRB2 critical for Angptl2 binding and activation. Blood 124(6):924–935
von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP et al (2007) The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Ann Intern Med 147(8):573–577
Liu J, Jackson CW, Gruppo RA, Jennings LK, Gartner TK (2005) The beta3 subunit of the integrin alphaIIbbeta3 regulates alphaIIb-mediated outside-in signaling. Blood 105(11):4345–4352
Yau JW, Singh KK, Hou Y, Lei X, Ramadan A, Quan A et al (2017) Endothelial-specific deletion of autophagy-related 7 (ATG7) attenuates arterial thrombosis in mice. J Thorac Cardiovasc Surg 154(3):978-988.e1
Wong C, Liu Y, Yip J, Chand R, Wee JL, Oates L et al (2009) CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo. Blood 113(8):1818–1828
Wall JE, Buijs-Wilts M, Arnold JT, Wang W, White MM, Jennings LK et al (1995) A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents. Br J Haematol 89(2):380–385
Szelenberger R, Kacprzak M, Bijak M, Saluk-Bijak J, Zielinska M (2019) Blood platelet surface receptor genetic variation and risk of thrombotic episodes. Clin Chim Acta 496:84–92
Williams AF, Barclay AN (1988) The immunoglobulin superfamily–domains for cell surface recognition. Annu Rev Immunol 6:381–405
Jandrot-Perrus M, Busfield S, Lagrue AH, Xiong X, Debili N, Chickering T et al (2000) Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily. Blood 96(5):1798–1807
Newman PJ, Newman DK (2003) Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology. Arterioscler Thromb Vasc Biol 23(6):953–964
Kang X, Cui C, Wang C, Wu G, Chen H, Lu Z et al (2018) CAMKs support development of acute myeloid leukemia. J Hematol Oncol 11(1):30
Locke D, Chen H, Liu Y, Liu C, Kahn ML (2002) Lipid rafts orchestrate signaling by the platelet receptor glycoprotein VI. J Biol Chem 277(21):18801–18809
Thomas SM, Brugge JS (1997) Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13:513–609
Pan G, Chang L, Zhang J, Liu Y, Hu L, Zhang S et al (2021) GSK669, a NOD2 receptor antagonist, inhibits thrombosis and oxidative stress via targeting platelet GPVI. Biochem Pharmacol 183:114315
Soriano Jerez EM, Gibbins JM, Hughes CE (2021) Targeting platelet inhibition receptors for novel therapies: PECAM-1 and G6b-B. Platelets 32(6):761–769
Parra-Izquierdo I, Melrose AR, Pang J, Lakshmanan HHS, Reitsma SE, Vavilapalli SH et al (2022) Janus kinase inhibitors ruxolitinib and baricitinib impair glycoprotein-VI mediated platelet function. Platelets 33(3):404–415
Babur Ö, Melrose AR, Cunliffe JM, Klimek J, Pang J, Sepp AI et al (2020) Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs. Blood 136(20):2346–2358
Gupta S, Cherpokova D, Spindler M, Morowski M, Bender M, Nieswandt B (2018) GPVI signaling is compromised in newly formed platelets after acute thrombocytopenia in mice. Blood 131(10):1106–1110
Ozaki Y, Suzuki-Inoue K, Inoue O (2013) Platelet receptors activated via mulitmerization: glycoprotein VI, GPIb-IX-V, and CLEC-2. J Thromb Haemost 11(Suppl 1):330–339
Liu Y, Hu M, Luo D, Yue M, Wang S, Chen X et al (2017) Class III PI3K positively regulates platelet activation and thrombosis via PI(3)P-directed function of NADPH oxidase. Arterioscler Thromb Vasc Biol 37(11):2075–2086
Watson SP, Auger JM, McCarty OJ, Pearce AC (2005) GPVI and integrin alphaIIb beta3 signaling in platelets. J Thromb Haemost 3(8):1752–1762
Billadeau DD, Leibson PJ (2002) ITAMs versus ITIMs: striking a balance during cell regulation. J Clin Invest 109(2):161–168
Moraes LA, Barrett NE, Jones CI, Holbrook LM, Spyridon M, Sage T et al (2010) Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells. J Thromb Haemost 8(11):2530–2541
Wee JL, Jackson DE (2005) The Ig-ITIM superfamily member PECAM-1 regulates the “outside-in” signaling properties of integrin alpha(IIb)beta3 in platelets. Blood 106(12):3816–3823
Mazharian A, Mori J, Wang YJ, Heising S, Neel BG, Watson SP et al (2013) Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function. Blood 121(20):4205–4220
Yip J, Alshahrani M, Beauchemin N, Jackson DE (2016) CEACAM1 regulates integrin αIIbβ3-mediated functions in platelets. Platelets 27(2):168–177
Alshahrani MM, Kyriacou RP, O’Malley CJ, Heinrich G, Najjar SM, Jackson DE (2016) CEACAM2 positively regulates integrin αIIbβ3-mediated platelet functions. Platelets 27(8):743–750
Maeda A, Kurosaki M, Ono M, Takai T, Kurosaki T (1998) Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal. J Exp Med 187(8):1355–1360
Ho LH, Uehara T, Chen CC, Kubagawa H, Cooper MD (1999) Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIR-B. Proc Natl Acad Sci USA 96(26):15086–15090
Acknowledgements
This work was supported by Chinese National Natural Science Foundation Grant (Grant No. 81970289, 82270340, 82300362), Experimental animal research project of Shanghai Science and Technology Innovation Action Plan (No. 22140901200), Cross Research Fund project of The Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Special Project of ShanghaiTech University, No. JYJC202127), SHIPM-mu fund from Shanghai Institute of Precision Medicine, Ninth People`s Hospital Shanghai Jiao Tong University School of Medicine (No. JC202005), Biological sample Bank project of The Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (No. YBKA202206), and the Clinical Research Program of 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine (No. JYLJ202014).
Funding
This work was supported by Chinese National Natural Science Foundation Grant (Grant No". 81970289, 82270340, 82300362), Experimental animal research project of Shanghai Science and Technology Innovation Action Plan (No. 22140901200), Cross Research Fund project of The Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Special Project of ShanghaiTech University, No. JYJC202127), SHIPM-mu fund from Shanghai Institute of Precision Medicine, Ninth People`s Hospital Shanghai Jiao Tong University School of Medicine (No. JC202005), Biological sample Bank project of The Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (No. YBKA202206), and the Clinical Research Program of 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine (No. JYLJ202014).
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Zhang Junfeng designed and guided this study. Zhang Tiantian, Zhang Mingliang and Guo Lingyu conducted the clinical study and all the experiments. Liu Dongsheng, Zhang Kandi, Bi Changlong and Zhang Peng participated in cell and animal experiments. Wang Jin, Fan Yuqi and He Qing participated in the clinical study. Alex C. Y. Chang guided the cell and animal experiments. All authors read and approved the final manuscript.
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All patients provided informed written consent prior to sample collection, and the study protocol was approved by the Shanghai Ninth People’s Hospital Institutional Ethics Committee (HKDL2017300) (No. 2016-256-T191) and performed in accordance with the ethical standards outlined in the 1964 World Medical Association Declaration of Helsinki. All animal experiments were approved by the Shanghai Ninth People’s Hospital institutional ethics committee (HKDL2017300).
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Zhang, T., Zhang, M., Guo, L. et al. Angiopoietin-like protein 2 inhibits thrombus formation. Mol Cell Biochem (2024). https://doi.org/10.1007/s11010-024-05034-9
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DOI: https://doi.org/10.1007/s11010-024-05034-9